Relationship between Toxoplasma gondii infection and psychiatric disorders in Iran: A systematic review with meta-analysis

Background Toxoplasma gondii, a ubiquitous parasitic protozoan, may be an important cause of neurological and psychiatric diseases. The present systematic review and meta-analysis, therefore, was conducted to investigate the scientific evidence regarding the potential association between T. gondii infection and psychiatric disorders in Iran. Methods We systematically reviewed articles from world-wide databases, including PubMed, Scopus, Science Direct, Web of Science, Google Scholar, and Iranian national databases up to July 30th, 2021. The Newcastle Ottawa Scale (NOS) was used to assess the quality of included studies. The common odds ratio (OR) was estimated using inverse variance and a random-effects model. Heterogeneity was assessed using the χ2-based Cochrane test (Q) and the I2 index. Also, sensitivity analyses and publication bias were calculated. Moreover, subgroup analysis was performed based on the type of disorder and quality score of different eligible studies. Results 16 studies were included in this meta-analysis. Our meta-analyses found that the OR of the risk of anti- T. gondii IgG and IgM in psychiatric patients compared to the control group was 1.56 (95% CI; 1.23–1.99) and 1.76 (95% CI: 1.19–2.61), respectively. Subgroup analysis based on the type of disorder showed that the OR of the risk of anti- T. gondii IgG in Iranian schizophrenia patients and other psychiatric disorders compared to the control group were 1.50 (95% CI; 1.09–2.07) and 2.03 (95% CI: 1.14–3.60), respectively, which are statistically significant. Also, the OR of the risk of anti- T. gondii IgM in Iranian schizophrenia and depression patients compared to the control group was 1.54 (95% CI; 0.9–2.64) and 1.03 (95% CI: 0.2–5.24), respectively, which are not statistically significant. Additionally, subgroup analysis based on quality scores showed no significant influence on the results according to the moderate quality studies. However, this association was significant according to the high quality studies. The obtained results of Egger’s test were 1.5 (95% CI; -0.62–3.73, P = 0.15) and 0.47 (95% CI; -0.82–1.76, P = 0.45), respectively, indicating publication bias. The significant results of the heterogeneity analysis confirmed a high level of heterogeneity in the IgG test (P = 0.000, I2 = 66.6%). However, no significant results from the test of heterogeneity were detected in the IgM test (P = 0.15, I2 = 27.5%). The results of the sensitivity analysis showed that the impact of each study on the meta-analysis was not significant on overall estimates. Conclusions Despite the limited number of studies, these outcomes supported a possible link between T. gondii infection and psychiatric disorders in Iran. However, more high-quality investigations are needed in the future.


Introduction
Toxoplasma gondii (T. gondii) is one of the most successful parasites in the world and is responsible for a neglected parasitic disease, toxoplasmosis, which forms tissue cysts in some tissues, including the brain of warm-blooded animals such as humans [1]. Human infection occurs in several ways, including by consumption of meat containing tissue cysts harboring bradyzoites and by ingestion of food or water contaminated with sporulated oocysts [2]. T. gondii prevalence in humans is different in developed and developing countries, and in some areas it can be high (e.g., Brazil, 77.5%; Sao Tome and Principe, 75.2%; Iran, 63.9%; Colombia, 63.5%; and Cuba, 61.8%) [3].
Tachyzoites, bradyzoites (the tissue cyst form), and sporozoites are three infectious forms of the T. gondii parasite. After the activation of the host's immune system, some of the tachyzoites differentiate into slowly-growing bradyzoite forms, establishing themselves within tissue cysts, which initiates the chronic or latent phase of the disease [4]. These tissue cysts can be reactivated when the immune function is weak [5].
Toxoplasmosis is commonly asymptomatic in immunocompetent individuals, although prenatal infection with this ubiquitous parasitic protozoan may cause abortion as well as a congenital disease that includes severe intellectual disability and seizures [6]. Also, different investigations suggested that chronic T. gondii infection could be related to various psychiatric disorders and changes in the personality of individuals in humans and animal models [7][8][9].
These disorders caused by T. gondii may be the result of the parasite's ability to infect a variety of brain cells, including neurons and microglial cells, causing death in host cells. In addition, IFN-is produced, which results in altered dopamine levels. Following T. gondii infection, a large amount of neurotransmitters, such as dopamine, are produced, which may be responsible for psychiatric disorders [10][11][12].
Results of previous systematic and meta-analysis studies have shown the association between T. gondii and some psychiatric disorders such as epilepsy, depression, bipolar disorder, and schizophrenia [13][14][15][16][17]. However, there has been no systematic review and meta-analysis describing the status of T. gondii and psychiatric disorders in Iran; hence, the current systematic review and meta-analysis was aimed to exploring the relationship between T. gondii and psychiatric disorders in this country.

Search strategy
This study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) and its checklist (S1 Checklist) [18]. In this systematic review and meta-analysis we sought to determine the epidemiological aspects of toxoplasmosis in patients with psychiatric disorders in Iran. The search strategy was performed based on the online literature screening in five English databases (PubMed, Scopus, Science Direct, Web of Science and Google Scholar) and three Persian databases (Magiran, Irandoc and Idml) up to July 30 th , 2021. This review was accomplished using medical subject heading (MeSH) terms in combination or alone: (Toxoplasma gondii OR Toxoplasmosis) AND (Mental Disorders OR Psychiatric Illness OR Psychiatric Disease OR Psychiatric Disorder OR Behavior Disorders) AND IRAN (S1 Table).

Inclusion and exclusion criteria
The inclusion criteria included: (1) studies published in English or Persian until July 30 th , 2021, (2) case-control and cross-sectional studies about the relationship between toxoplasmosis and psychiatric disorders, (3) original research papers, (4) original articles with available full texts in English and Persian languages, and (5) studies with information on the exact total sample size and positive samples in the case and control groups, (6) studies performed only on humans, (7) studies providing detailed information on the prevalence of toxoplasmosis using serology (i.e., IgG and/or IgM antibodies). The exclusion criteria were: (1) studies in other countries, expect Iran, (2) review articles, and (3) non-human studies.

Study selection and data extraction
All the retrieved papers from the search strategy were imported to EndNote (version X7). After the removal of duplicate papers, the titles and abstracts were studied exactly by two different reviewers (EM and MM). The reviews, systematic review and meta-analysis, studies in other countries, non-serological studies, and animal experimental studies were excluded to assess the final eligibility and inclusion criteria. Then, the information about the name of the first author, year of publication, psychosomatic disorder, location of the study, type of study, method, type of antibody, and age and gender of Iranian patients with psychiatric disorders and controls was extracted into a Microsoft Excel datasheet.

Quality assessment
In order to assess the quality of reporting of the studies, the Newcastle Ottawa Scale (NOS) with separate criteria for case-control and cross-sectional studies was used [19]. Based on the NOS checklist, in the case-control studies, the scores of 3, 4-6, and 7-9 were representative of low, moderate, and high quality, respectively. Furthermore, regarding the cross-sectional studies, the scores of 1-2, 3-5, and 6-7 indicated low, moderate, and high quality, respectively.

Meta-analysis
The meta-analysis was executed with STATA version 11 (STATA Corp., College Station, Texas, USA). The common odds ratio (OR) was estimated using inverse variance and a random-effects model for each included study. To display the heterogeneity between studies, χ2-based Cochrane test (Q) and I 2 index were applied [20]. The publication bias was examined by the Egger test [21]. Also, a sensitivity analysis was performed to identify the probable effect of each article on the overall results by excluding them using STATA software. Additionally, subgroup analysis was performed based on the type of disorder and quality score of different eligible studies.

Results
Full texts of 20 articles had eligibility to be accounted for in the systematic review according to inclusion criteria, as shown in Fig 1. Out of 20 studies, a total of 4 papers [22][23][24][25] were excluded due to the lack of a control group, and eventually, 16 of these articles (two studies containing 2 datasets and one study containing 4 datasets) were entered into this meta-analysis with respect to the inclusion/exclusion criteria. Information and characteristics of each study in systematic review and meta-analysis are presented in Table 1 and S2 Table, respectively. The included studies in the present meta-analysis showed acceptable quality (i.e., �3 for crosssectional studies and �4 for case-control studies). S3 Table shows the quality of the included studies.
The enzyme-linked immunosorbent assay (ELISA) was the most commonly used method to detect anti-T. gondii IgG and IgM antibodies in studies. 7 and 13 studies addressed anti-T. gondii IgG, and both IgG and IgM, respectively.

PLOS ONE
Toxoplasma gondii and psychosomatic disorders The results of the meta-analysis indicate that the OR of the risk of anti-T. gondii IgG and IgM in psychiatric patients compared to the control group was 1.56 (95% CI; 1.23-1.99) and 1.76 (95% CI: 1.19-2.61), respectively, which are statistically significant (Figs 2 and 3).
Subgroup analysis based on the type of disorder showed that the OR of the risk of anti-T. gondii IgG in Iranian schizophrenia patients and other psychiatric disorders compared to the control group were 1.50 (95% CI; 1.09-2.07) and 2.03 (95% CI: 1.14-3.60), respectively, which are statistically significant (Fig 4). Also, the OR of the risk of anti-T. gondii IgM in Iranian schizophrenia and depression patients compared to the control group was 1.54 (95% CI; 0.9-2.64) and 1.03 (95% CI: 0.2-5.24), respectively, which are not statistically significant (Fig 5). Additionally, subgroup analysis based on quality scores of different eligible studies showed that the OR of the risk of anti-T. gondii IgG in other psychiatric patients compared to the control group was 1.68 (95% CI; 1.28-2.20) and 1.43 (95% CI; 0.91-2.27), respectively (Fig 6). Also, the OR of the risk of anti-T. gondii IgM in psychiatric patients compared to the control group based on the high and moderate quality studies were 2.58 (95% CI; 1.57-4.23) and 1.22 (95% CI; 0.75-1.97), respectively (Fig 7).
The results of sensitivity analysis showed that the impact of each study on the meta-analysis was not significant on overall estimates (Figs 10 and 11).

Discussion
Nowadays, investigation on the correlation between T. gondii infection and psychiatric disorders has become a hot topic [26]. This systematic review and meta-analysis aimed to quantify the pooled ORs of anti-T. gondii IgG and IgM antibodies in Iranian psychiatric patients and compare them with those of control groups in Iran. The results of the present study indicated a significantly higher T. gondii IgG and IgM in the psychiatric patients compared to those in the control group, with an OR of 1.56 (95% CI; 1.23-1.99) and 1.45 (95% CI: 1.10-1.92). Based on these results, toxoplasmosis can be considered a risk factor for psychiatric patients, and thus, these patients should pay attention to the detection of T. gondii in this area. Based on subgroup analysis, the OR of the risk of anti-T. gondii IgG in Iranian schizophrenia and other psychiatric disorder patients was > 1 and significant. However, this connection between latent toxoplasmosis and depression was not significant. Given that few studies have reported a link between certain psychiatric disorders (bipolar, mental, anxiety, hyperactivity, and intellectual disability) and Toxoplasma infection in Iran, a subgroup analysis based on the type of disorder was performed in tandem. In addition, the small number of studies limits the reliability of results, and more investigation are required to shed more light on this line. Similar to our results, several meta-analysis studies have reported a positive association between T.

PLOS ONE
gondii infection with a higher IgG level and the presence of schizophrenia [14][15][16]27]. Thus, T. gondii infection can be considered an underlying component of the pathophysiology of schizophrenia [26]. Also, similar to our results a meta-analysis by Cossu et al. which consisted of a total of 4021 patients with bipolar disorder and 8669 healthy control people in 23 studies, recently revealed that the prevalence of IgG class antibodies to T. gondii in patients with bipolar disorder was significantly higher than the prevalence of antibodies in control groups [28].
Contradictory results have been reported regarding the possible link between T. gondii infection and depression. However, despite the limited number of studies, a relationship between T. gondii seropositivity and depression cannot be confirmed. Our findings are consistent with the findings of the two previous meta-analyses [14,29].
According to our subgroup analysis, a significant association of T. gondii IgM antibodies and Iranian schizophrenia and depression patients was not observed. Based on these results, acute toxoplasmosis is not considered a risk factor for these patients. However, the prevalence of T. gondii IgM in patients with other psychiatric disorders was significantly higher than the prevalence of IgM in control groups, with a combined OR of 2.76 (95% CI: 1. 41-5.39). In the meta-analysis performed by Sutterland et al., on the association of T. gondii infection with recent onset schizophrenia, a non-significant OR of 1.24 (95% CI 0.98-1.57, P = 0.08) was

PLOS ONE
found [14]. In return, Monroe et al., in a meta-analysis reported a significant increase in the risk of positive T. gondii IgM antibodies in acute psychosis in patients with schizophrenia compared with controls [30]. IgM is an indicator of an acute or recent infection or a potentially persistent infection or reinfection, probably with a different genotype of T. gondii [31][32][33][34]. These variations in these findings might be related to sociocultural habits, geographical and environmental factors, sample size, and methodology in the studied population [35][36][37]. Moreover, there is evidence that different genotypes of T. gondii have diverse effects on the course of psychosis [38]. The study of these variables aid in improving the precision of future studies describing the relationship between infectious agents and psychiatric disorders [39]. However, due to different age ranges and gender proportions of participants and the almost identical methods in the selected studies (ELISA), subgroup analysis based on these two variables was not performed in this study. These variables may have affected the outcomes of our meta-analysis. Additionally, we found no significant influence on the results from subgroup analysis according to the moderate quality studies based on anti-T. gondii IgG and IgM in psychiatric patients. However, this association was significant according to the high quality studies. A higher score on the NOS checklist indicates a higher quality of a study. In fact, the quality of the majority of the effective items was taken into account in these studies.

PLOS ONE
Furthermore, we found high heterogeneity in the association between psychiatric disorders and T. gondii infection in this study. There are several sources of heterogeneity. In case-control studies, selection of cases and controls in different populations and in different ways has been done. Moreover, different ways of selecting the target groups can be a source of heterogeneity. The age of the participants in the studies is another source of heterogeneity because advanced age increases the chance of exposure to Toxoplasma.
The included studies in our meta-analysis study were from 10 different cities in Iran. However, data gaps were identified for other cities where no data was available on the association between T. gondii and psychiatric disorders. Therefore, it is highly necessary to find experimental documents from other parts of Iran.

Conclusions
In conclusion, to the best of our knowledge, this is the first systematic review and meta-analysis providing a general view of a possible link between T. gondii and psychiatric disorders in Iran. However, many questions remained to be answered in future investigations. Also, further research should be performed to investigate into measures that could decrease the high prevalence of Toxoplasma in Iran.